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Single-copy locus proteomics of early- and late-firing DNA replication origins identifies a role of Ask1/DASH complex in replication timing control.

Published:28 February 2023 DOI: 10.1016/j.celrep.2023.112045 PMID: 36701236
Matthias Weiβ, Anna Chanou, Tamas Schauer, Andrey Tvardovskiy, Stefan Meiser, Ann-Christine K?nig, Tobias Schmidt, Elisabeth Kruse, Henning Ummethum, Manuel Trauner, Marcel Werner, Maxime Lalonde, Stefanie M Hauck, Antonio Scialdone, Stephan Hamperl

Abstract

The chromatin environment at origins of replication is thought to influence DNA replication initiation in eukaryotic genomes. However, it remains unclear how and which chromatin features control the firing of early-efficient (EE) or late-inefficient (LI) origins. Here, we use site-specific recombination and single-locus chromatin isolation to purify EE and LI replication origins in Saccharomyces cerevisiae. Using mass spectrometry, we define the protein composition of native chromatin regions surrounding the EE and LI replication start sites. In addition to known origin interactors, we find the microtubule-binding Ask1/DASH complex as an origin-regulating factor. Strikingly, tethering of Ask1 to individual origin sites advances replication timing (RT) of the targeted chromosomal domain. Targeted degradation of Ask1 globally changes RT of a subset of origins, which can be reproduced by inhibiting microtubule dynamics. Thus, our findings mechanistically connect RT and chromosomal organization via Ask1/DASH with the microtubule cytoskeleton.

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